Project Summary Proper blood cell formation by rare, self-renewing hematopoietic stem cells (HSCs) in the bone marrow (BM) results from a complex series of fate decisions that are controlled via an array of genetic, epigenetic and metabolic programs. Notably, mutations in genes regulating these programs often lead to HSC dysfunction and/or the development of hematological malignancies. Recently, somatic mutations in pre-RNA splicing factors such as U2AF1 have been observed in hematopoietic cells of patients with myelodysplastic syndrome (MDS), a heterogeneous and difficult-to-treat clonal blood disorder characterized by cytopenias in one or more blood lineages and the potential to evolve into acute myelogenous leukemia (AML). Notably, U2AF1 mutations are considered early events in MDS pathogenesis, suggesting that aberrant pre-RNA splicing in HSCs drives the evolution to disease by deregulating HSC fate. However, the role of U2AF1 in regulating HSC function in normal hematopoiesis, and the contribution of mutant U2AF1 to disease is unclear. Emerging evidence suggests that MDS, AML and other hematological malignancies are associated with prior or concurrent onset of chronic inflammatory diseases including rheumatoid arthritis, lupus, and obesity, and indeed MDS itself is typically characterized by overproduction of inflammatory cytokines and hyperactive innate immune signaling. Moreover, inflammatory signals such as interleukin-1 (IL-1) can directly impact HSC fate, leading to overproduction of myeloid cells at the expense of self-renewal and balanced lineage output. Strikingly, U2AF1 has been shown to play an important role in regulating cellular responses to inflammation by generating anti-inflammatory splice forms of key immune signaling genes such as MyD88. Thus, we hypothesize that U2AF1 plays a critical role in regulating HSC responses to inflammatory signals, with aberrant splicing leading to hyperactive responses to inflammatory signals, resulting in a positive feedback loop that promotes deregulated blood production and altered HSC self-renewal. We propose that aberrant mRNA splicing and inflammation are therefore mechanistically linked and are crucial drivers of hematopoietic dysfunction and, potentially, evolution to MDS and other blood disorders. This proposal seeks to identify how pre-RNA splicing, and U2AF1 in particular, regulates the self-renewal and differentiation of HSCs in the context of inflammation using a combination of in vitro assays and in vivo mouse models of chronic inflammation using WT and U2AF1S34F transgenic mice. These investigations will fill a significant gap in our understanding of how aberrant splicing promote hematopoietic dysfunction and eventual progression to malignancy, by investigating whether aberrant splicing in U2AF1 mutant HSCs exacerbates their response to inflammatory signaling. Collectively, they will provide needed insight into the role of U2AF1 in regulating HSC responses to inflammation and stand to identify a previously unexplored link between aberrant pre-RNA splicing, chronic inflammation, and the pathogenesis of complex blood disorders such as MDS.